1. Field of the Invention
The present invention relates to a method for determining the activity of a cell cycle regulatory factor without using a radioisotope and a method for diagnosing a cancer using the method.
2. Description of Related Art
Cell proliferation is a fundamental and important feature of living things. The cell proliferation involves division of a single cell into two daughter cells, and somatic cells divide through a plurality of sequential reactions including growth of cells, replication of DNAs, distribution of chromosomes and division of cells. This chain of sequential reactions is referred to as a cell cycle. In the case of eucaryotic cells, the cell cycle is divided into four phases, that is, a synthetic (S) phase during which the replication of DNAs takes place, a mitotic (M) phase during which the division of cells takes place, a gap 1 (G1) phase which is an interphase from the M phase to the next S phase and a gap 2 (G2) phase which is an interphase from the S phase to the next M phase. In the G1 phase, cells receive a signal for proliferation, prepare for the replication of DNAs, and make metabolism and growth which are necessary for the division of cells. In the G2 phase, the cells prepare for the division. In the G1 phase, a transit point is experimentally assumed which is called an R point (restriction point) for mammalian cells and START for yeast. Typically, cells multiply in response to proliferation signals from the outside. The cells receive the signals in the G1 phase and progress the cell cycle. After passing through a certain point in the G1 phase, the cell cycle progresses from the S phase to the G2 phase, the M phase and then the G1 phase without stopping even if the proliferation signals are not received any longer. This certain point is the R point or START, and is a so-to-speak point at which the entering into cell cycle progression is determined. Further, the cells can leave the cell cycle and enter a resting (G0) phase during which the cell do not grow or multiply. Experimentally, the cells entering the resting phase, if given a suitable signal, can be returned to the G1 phase and induced to grow and divide again. It is considered that a lot of non-growing and non-multiplying cells of multicellular organisms are in the G0 phase.
There mainly exist two groups of cell cycle regulatory factors in cells. One is a group of kinases which are positive regulatory factors and are referred to as cyclin-dependent kinases (CDKs), and the other is a group of CDK inhibitors (CDKIs) which are negative regulatory factors. The CDKs exist in cytoplasm in the inactive form. The CDKs are activated, e.g., by phosphorylation, and move into nuclei in the cells. In the nuclei, the CDKs bind to cyclin molecules to form complexes with cyclin (referred to as activated CDKs hereinafter) and positively regulate the progress of the cell cycle at various steps of the cell cycle. On the other hand, the CDKIs inactivate the CDKs by binding to the activated CDKs or CDK simple substances, thereby regulating the cell cycle negatively.
There are now known seven types of CDKs, i.e., CDK1, CDK2, CDK3, CDK4, CDK5, CDK6 and CDK7 to which different cyclins are bound. More particularly, CDK1 binds to cyclin A or B, CDK2 binds to cyclin A or E, and CDK4 and CDK6 bind to cyclin D1, D2 or D3, to be activated. The activated CDKs control specific phases of the cell cycle. The following table 1 shows CDKs concerning the control of the cell cycle, cyclins which functionally bind to the CDKs, and phases of the cell cycle during which the activated CDKs act.
TABLE 1Cyclins binding toPhases of cell cycle inCDKsCDKswhich activated CDKs actCDK4, CDK6Cyclin D1, D2, D3G1CDK2Cyclin ETransitional periodfrom G1 to SCDK2Cyclin ASCDK1Cyclin A, BTransitional periodfrom S to M, M
Thus the cell cycle is controlled and the cell proliferation is regulated by activation of different types of CDKs. The activated CDKs are enzymes which phosphorylate serine residue and threonine residue in a protein as a substrate. In an in-vitro reaction system, the activated CDK1 and CDK2 react well on histone H1 as a substrate and the activated CDK 4 and CDK6 react well on Rb (retinoblastoma protein) as a substrate. In an in-vivo cell cycle regulation, it is considered that the activated CDKs require Rb as a physiologic substrate, but it is not known what other proteins act as substrates.
As described above, the CDKs and cyclins regulate the cell cycle in close association with each other. The multiplication of cyclin D1 gene is observed in a great number of cases of esophageal cancer, while over expression of cyclin D1 gene is observed in a great number of cases of stomach cancer and colon cancer. On the other hand, the multiplication of cyclin E gene is observed in stomach cancer and colon cancer but is not observed in esophageal cancer. Excessive expression of cyclin E in stomach and large bowel takes place with great frequency in cases of adenoma and adenocarcinoma and shows a significant correlation with malignancy such as invasion, progress of stages, metastasis and the like. The expression and kinase activity of CDK1 are remarkably accelerated in most cases of stomach cancer and colon cancer as compared with normal mucosal tissue. It is known that augmented expression of cyclin genes correlates with the progress and malignancy of various cancers (see Wataru Yasui, Sysmex Journal Web., p. 1 to p. 10, vol. 1, 2000).
Therefore, it is expected that measuring the activity of the individual species of CDKs will provide good indices of the type and malignancy of diseases related to the control of the cell cycle. In other words, generally at the R point, the expression of CDK2 decreases and the cell cycle arrest and the division of cells is controlled. However, if the expression of CDK2 increases at the R point, it means that the cell cycle fails to stop, i.e., it means a state of a disease such as cancer. If the expression of CDK4 or CDK6 increases, stomach cancer or colon cancer may be expected because stomach cancer and colon cancer involve accelerated gene expression of the cyclin D1 which bind specifically to CDK4 or CDK6. Thus, it is considered to be possible to determine the type of cancer.
Usually, the activity of the CDKs is determined using radioisotopes. More particularly, in the presence of a CDK which is extracted from a cell lysate by an immunoprecipitation method using an anti-CDK antibody and whose activity is unknown, 32P-labelled adenosine 5′-O-(3-triphosphate) (ATP) is reacted with serine residue or threonine residue in a substrate to introduce monophosphate group derived from the 32P-labelled ATP. The amount of 32P taken by the substrate is detected by autoradiography or by a scintillation counter. Thereby the amount of the phosphorylated substrate is measured and the activity of the CDK is calculated from the amount of the phosphorylated substrate.
This method requires careful attention in handling the substance and in disposal of waste liquid since it uses 32P which is a radioisotope.